Hi Everyone,
I am trying to do some work on G-protiens and need the following question answered...

It deals with embryonic stem cells that have a temperature sensitive mutation in the G alpha 's' protien. This G-protien is locked on at 33 degrees celcius and locked off at 37 degrees celcius. At the 37 degrees, in vitro, the cells remain undifferentiated (self renewal) in the presence of a cytokine (LIF) but they differentiate into epithelial cells forming tubular structures in the presence of HGF (a growth factor) at the same temperatute, 37 degrees celcius. When the cells are exposed to both LIF and HGF at 37C, the cells undergo self renewal ONLY. WHen you repeat the above experiments at 33C, the cells differentiate in ALL CASES.
So what I need is to describe the signalling cascades and the interactions between them that explains the results above. Also, I need to come up with an experimental approach to test for these interactions.
I'd REALLY REALLY appreciate ANYTHING ANYONE can contribute to this...THANKS!!!!!

cellularbiostudent2 wrote:Hi Everyone, I am trying to do some work on G-protiens and need the following question answered...

It deals with embryonic stem cells that have a temperature sensitive mutation in the G alpha 's' protien. This G-protien is locked on at 33 degrees celcius and locked off at 37 degrees celcius. At the 37 degrees, in vitro, the cells remain undifferentiated (self renewal) in the presence of a cytokine (LIF) but they differentiate into epithelial cells forming tubular structures in the presence of HGF (a growth factor) at the same temperatute, 37 degrees celcius. When the cells are exposed to both LIF and HGF at 37C, the cells undergo self renewal ONLY. WHen you repeat the above experiments at 33C, the cells differentiate in ALL CASES. So what I need is to describe the signalling cascades and the interactions between them that explains the results above. Also, I need to come up with an experimental approach to test for these interactions.I'd REALLY REALLY appreciate ANYTHING ANYONE can contribute to this...THANKS!!!!!

Hi, .
I think, you should do some more experiments, if possible.. You may try the following -
-- try some experiments in order to know the nature of those tubular structures , this may give you an idea aboout what is happenig.
-- try these expt.s using different agents [ cytokines and growth factors] but, you should know the similarities and differences [ these can be structural, in cascades etc.] between these agents in order to predict the result . If the predicted result matches with the actual one then your prediction may be correct - then test it. But if it doesnot match then this will help you decide what to next.If there are some agents that join the same cascade at later step than these ? If yes the try them , this may give you an idea about which part/s of the cascade is
affected.
Though this is a case of mutation &we know that there is change in G alpha s proteins but there are more than one phenomena affected by G alpha s proteins, so this will help you know what is really affected.

There are several different classifications of receptors that couple signal transduction to G-proteins. These classes of receptor are termed G-protein coupled receptors, GPCRs. Well over 1000 different GPCRs have been cloned, most being orphan receptors having no as yet identified ligand. Three different classes of GPCR are reviewed:
1. GPCRs that modulate adenylate cyclase activity. One class of adenylate cyclase modulating receptors activate the enzyme leading to the production of cAMP as the second messenger. Receptors of this class include the b-adrenergic, glucagon and odorant molecule receptors. Increases in the production of cAMP leads to an increase in the activity of PKA in the case of b-adrenergic and glucagon receptors. In the case of odorant molecule receptors the increase in cAMP leads to the activation of ion channels. In contrast to increased adenylate cyclase activity, the a-type adrenergic receptors are coupled to inhibitory G-proteins that repress adenylate cyclase activity upon receptor activation.
2. GPCRs that activate PLC-g leading to hydrolysis of polyphosphoinositides (e.g. PIP2) generating the second messengers, diacylglycerol (DAG) and inositoltrisphosphate (IP3). This class of receptors includes the angiotensin, bradykinin and vasopressin receptors.
3. A novel class of GPCRs are the photoreceptors. This class is coupled to a G-protein termed transducin that activates a phosphodiesterase which leads to a decrease in the level of cGMP. The drop in cGMP then results in the closing of a Na+/Ca2+ channel leading to hyperpolarization of the cell. See the Role of Vitamin A in Vision for more details.

G-Protein Regulators

The activity of G-proteins with respect to GTP hydrolysis is regulated by a family of proteins termed GTPase activating proteins, GAPs. The proto-oncogenic protein, Ras, is a G-protein involved in the genesis of numerous forms of cancer (when the protein sustains specific mutations). Of particular clinical significance is the fact that oncogenic activation of Ras occurs with higher frequency than any other gene in the development of colo-rectal cancers. Regulation of Ras GTPase activity is controlled by rasGAP.
There are several other GAP proteins besides rasGAP that are important in signal transduction. There are two clinically important proteins of the GAP family of proteins. One is the gene product of the neurofibromatosis type-1 (NF1) susceptibility locus. The NF1 gene is a tumor suppressor gene and the protein encoded is called neurofibromin. The second is the protein encoded by the BCR locus (break point cluster region gene). The BCR locus is rearranged in the Philadelphia+ chromosome (Ph+) observed with high frequency in chronic myelogenous leukemias (CMLs) and acute lymphocytic leukemias (ALLs).